Progressive resistance training (PRE) is recommended for adolescents to improve their health, fitness, and strength (6). Unlike in adults, in adolescents, strength gains occur as a normal course of growth and maturation. Muscle growth is rapid during adolescence because muscle fibers increase almost double in diameter (24). In addition, with adequate PRE, strength gains in adolescents may occur above normal development (6). Strength gains during adolescence correlate with an increase in muscle fiber size, but it is assumed that PRE-related gains in strength (∼30% gains in 8-week programs) are largely because of neurological adaptation rather than because of an increase in muscle cross-sectional area (CSA) or hypertrophy (26). However, studies of muscle hypertrophy in human adolescents have been imprecise, and therefore, in adolescents, it is unclear if PRE elicits hypertrophy of muscle above and beyond normal muscle growth. It is possible that strength gains from moderate intensity PRE prescribed for adolescents may not be distinguishable from normal growth, especially in terms of muscle hypertrophy. Interestingly in human adolescents, moderate loading may actually be more advantageous than heavy loading in terms of strength gains and safety (7).
Progressive resistance training interventions in adolescent rodents have provided more precise investigations of muscle adaptation. Data from rodents have shown that hypertrophy of muscle does occur with PRE in adolescent animals with heavy loading regimens. Using a climbing apparatus and tail weights, Yarasheski et al. (31) reported an approximately 15% increase in fiber CSA across muscles after an 8-week PRE program in 3-week-old rats that achieved loads of up to 250% above the animal body weight. It is noteworthy that these findings were similar in different fiber types, with an approximately 13% increase in the CSA for rectus femoris fibers classified as type IIb (expressing myosin heavy chain 2B [MyHC2B]) and an approximately 16% increase in type IIa fibers (expressing MyHC2A) in the PRE animals compared with that in controls. Duncan et al. (5) implemented a progressive PRE protocol for 26 weeks using 3-week-old rats, and these rats progressed to carrying loads equivalent to 140% of their final body weight. The CSA across muscle fibers was reported to increase ranging from 21 to 59% gains between the various muscle groups investigated. It is important to recognize that these rodent studies used heavy loading, which may not be representative of more moderate loads recommended for human adolescents. The effect of more moderate loading on muscle hypertrophy in adolescent rodents has not been explored.
The mechanism by which resistance training stimulates hypertrophy in adolescents is presumed to occur via endogenous growth factors because testosterone levels are already substantially increased during this time (26). In adults, several growth factor–dependent signaling proteins have been observed to become phosphorylated within 24 hours of resistance exercise including mammalian target of rapamycin (mTOR), Akt, p70S6 kinase, and ribosomal protein S6 (rpS6), and the phosphorylation of these molecules correlates with increases in muscle protein synthesis and hypertrophy (3,17,29). After 8 weeks of human strength training, mTOR mRNA levels elevate suggesting that mTOR protein levels may become chronically elevated as well (16). The significantly greater growth rates occurring in adolescents may elicit different signaling mechanisms in response to PRE compared with that in adults. Because muscles actively grow during adolescence, it is possible that all (or some) of these signaling pathways are already maximally activated. Exercise-induced muscle hypertrophy in adolescents could involve increased expression and more sustained activation of these pathways compared with that in adults. The interaction between training regimens and adolescent growth has not been explored with respect to this. Relatively little has been reported on PRE in adolescents with regard to the mechanisms of muscle adaptation. Specifically, it is presently unknown whether exercise in adolescents results in sustained elevations in the phosphorylation levels of signaling molecules such as mTOR, Akt, p70S6 Kinase, or rp S6 and increased expression of these proteins.
The purpose of this study was to investigate whether moderate PRE stimulates muscle hypertrophy in adolescent animals and to concurrently investigate the effects of PRE on mTOR, Akt, and rpS6 protein expression and phosphorylation. The rat flexor hallucis longus (FHL) muscle was examined because it was previously observed to hypertrophy after PRE in adults (12). Consistent with the expected muscle hypertrophy, we hypothesized that a 10-week PRE regimen starting at 3 weeks postnatally would increase (a) fiber CSA and (b) MyHC content. In addition, we hypothesized that the expression and phosphorylation levels of mTOR, Akt, and rpS6 will increase after 10 weeks of PRE compared with that in sedentary (SED) controls of a similar age.
Experimental Approach to the Problem
We hypothesized that moderate intensity PRE in adolescent animals (up to 80% body weight) would stimulate muscle hypertrophy (i.e., an increase in fiber CSA) in a growth factor–dependent manner. To precisely investigate muscle CSA and to obtain biochemical information regarding MyHC expression and growth factor signaling, we used young adolescent rodents. Although several animal models (e.g., hindlimb unloading or synergist ablation) produce robust muscle hypertrophy in remaining muscles, these models preclude analyses of normal adolescent growth. The PRE is a training regimen in which exercise intensity is optimized by gradually increasing muscle load over time, and it induces muscle hypertrophy within 6–8 weeks (6–12 repetition maximums with multiple sets, 2–3 d·wk−1) (1), making PRE a useful model for studies of the interaction between resistance training and adolescent muscle growth. In this study, we used a ladder-climbing apparatus and gradually added mass to the tails of rats over time to simulate a PRE program comparable with that in humans as used by other investigators (5,12,31). Control animals were SED animals that were caged in exactly the same manner as that of the PRE group and were not provided the PRE training. Comparisons were made between groups to establish changes in muscle CSA, MyHC expression, and changes in growth factor–dependent signaling molecules.
All the experimental procedures were approved by the Mayo Clinic Institutional Animal Care and Use Committee. Mayo Clinic conducts research in a manner consistent with the guidelines of the National Institutes of Health (NIH; #A3291-01), United States Department of Agriculture (#41-R-0006), and AAALAC (#000717). Sixteen, 3-week-old (∼56 g), male Sprague-Dawley rats (Harlan Laboratories, Madison, WI, USA) were randomly assigned to either a PRE trained group or an SED group. Both groups of rats were allowed free access to food and water. The animals were housed in a 12:12 light-dark cycle environment with stable room temperature (22°C). The PRE rats were removed from the housing room 3 times per week to be trained and were trained at the same time each day to minimize variability. The SED animals were also removed from the housing room, but they remained in their cages for the duration of the training. Both groups of animals were weighed weekly.
Animal Training Protocol
Three-week old, male Sprague-Dawley rats (55.3 ± 1.1 g) were trained to climb a vertical ladder as a mode for PRE. Trained animals began climbing with a body-weight-only load, and the external load was progressively increased each week such that at the end of 10 weeks, all the animals in the PRE group had progressed to carry an additional 80% of body weight per climb (Figure 1B). This resistance workload is equivalent to approximately 3 J at week 1 and approximately 24 J per exercise session at week 10. Both SED and PRE were free fed and increased in body weight during the 10-week study period, consistent with expected postnatal growth. The PRE training protocol used a vertical ladder (40 cm long and 30 cm wide) comprising 54 wooden rungs (5 mm in diameter) with a platform at the top. The rats in the PRE group had a 50-ml conical tube taped to the base of their tail (containing a variable mass of steel shot) and were placed on the bottom rung of the ladder allowing them to climb to the top platform (Figure 1A) 3 times per week. The training protocol consisted of 3 sets of 10 repetitions with 2 minutes of rest between sets. The rats that did not successfully climb to the platform in <10 seconds were placed on the bottom rung, and if a rat refused to climb for 3 consecutive attempts, the exercise set was terminated. The resistance workload was increased approximately10% each week (by increasing the mass of the steel shot) only if the rat was able to complete all 30 repetitions.
The FHL muscle was removed from the animals 24 hours after the final training session (17). The animals were anesthetized with xylazine (10 mg·kg−1) and ketamine (90 mg·kg−1), and the entire FHL muscle was dissected from the flexor digitorum longus and posterior tibialis muscles of the posterolateral leg on each side. One FHL muscle was quickly frozen for protein analyses, whereas the other was processed for histochemical analyses after freezing at optimal length in melting isopentane.
Muscle Fiber Type Proportions and Cross-Sectional Area
Serial cross sections from the midportion of each muscle were cut at 10-μm thickness with a cryostat (Reichert-Jung, model 2800E, Nassloch, Germany). The sections were reacted with mouse primary antibodies against MyHC isoforms as previously reported (19,30,33). Antibodies against MyHCSlow (IgG, Novocastra, Nassloch, Germany), MyHC2A (IgG, hybridoma—Blau A4.74), MyHC2B (IgM, hybridoma—Schiaffino BFF3), and BF-35 antibody (IgG, hybridoma—which reacts with all MyHC isoforms but MyHC2X) were diluted 1:200 in phosphate-buffered saline (PBS) containing 0.5% bovine serum albumin (5 mg·ml−1). Muscle sections were incubated in primary antibody for approximately 2 hours at room temperature, washed in PBS, and reacted with Cy3-conjugated secondary antibodies (goat antimouse IgG or goat antimouse IgM) for 1 hour. The sections incubated with only the secondary antibodies served as controls for nonspecific reactivity of all secondary antibodies (10). The slides were imaged with an Olympus Fluoview confocal microscope mounted on a BX50WI microscope (Olympus America, Melville, NY, USA) using a green HeNe laser (543 nm). Representative sections were imaged with an Olympus DApo 40x/1.3 NA oil immersion objective in arrays of 800 × 600 pixels (400 × 300 μm). Fiber type proportions and CSA measurements were performed in at least 8 sections per muscle. All the fibers in the image were analyzed such that approximately 100 fibers were fiber type identified per muscle. Fiber type proportions were determined directly using the total number of fibers of each type in all the sections obtained from a muscle.
Myosin Heavy Chain Content
The procedure for myosin protein determination from skeletal muscle was described previously (8–10). Basically, MyHC isoforms were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). Known concentrations of purified rabbit myosin (Sigma, St. Louis, MO, USA), verified with the BioRad-DC kit according to the manufacturer's protocol, were run in parallel to permit the quantification of MyHC in FHL samples. The expression of different MyHC isoforms was quantified by densitometry. The MyHC content was then normalized by tissue mass.
Protein Expression and Phosphorylation of Translational Regulators
We examined signal transduction proteins that are positive regulators of protein translation (2,13,22,25) including mTOR, Akt, and S6 ribosomal protein (rpS6). We hypothesized that each of these signaling proteins would have a higher expression and greater phosphorylation levels (of critical amino acid motifs) in PRE compared with that in the SED animals. We also examined AMP kinase (AMPK), a reported antagonist of mTOR signaling (15). We hypothesized that the expression and phosphorylation of AMPK would not be different between groups. Two hundred milligrams of FHL was lysed on ice using a glass mortar and pestle and diluted in 1 ml of cell lysis buffer (in millimoles per liter: Tris-HCl [pH 7.5] 20, NaCl 150, disodium salt of ethylenediaminetetraacetic acid 1, ethylene glycol tetraacetic acid 1, Na4P2O7 2.5, β-glycerophosphate 1, Na3VO4 1, and 1% Triton) containing 1 μg·mL−1 leupeptin and phosphatase inhibitor cocktail (Roche Applied Science, Indianapolis, IN, USA). Total protein concentration was determined using the BioRad Dc Assay, and 200 μg of protein was loaded per well on either 7 or 15% BioRad Criterion SDS-PAGE gels. Proteins were resolved by gel electrophoresis and transferred to poly(vinylidene fluoride) membranes as described by the manufacturer (BioRad Criterion Gel System). The membranes were blocked in 5% milk in 1× Tris-buffered saline (TBS; pH = 7.4, Biorad, Hercules, CA, USA) for 30 minutes and incubated overnight at 4°C with primary antibody. Primary antibodies were used at a dilution of 1:1,000 in 5% milk in 1× TBS (all from Cell Technology Inc., Beverly, MA, USA) and included both phosphorylated and total Akt (#9271/9272), 5 AMP-activated protein kinase (#2603/2535), mammalian target of rapamycin (#2971/2974), and S6 ribosomal proteins (#4858/2217). The membranes were then washed 3 times with TBS for 5 minutes and probed with antirabbit horse radish peroxidase–conjugated secondary antibody in TBS for 1 hour. The membranes were washed 3 times with TBS for 5 minutes, and protein was detected using Super Signal Dura enhanced chemiluminescence as described by the manufacturer (Pierce, Thermo Fisher Scientific, Waltham, MA, USA). The membranes were probed and stripped serially for phosphorylated forms and the total of each of the signaling proteins as described above. Restore Western Blot Stripping Buffer (Pierce, Thermo Fisher Scientific) was used according to the manufacturer's instructions to remove previously applied antibodies. The membranes were also probed for β-tubulin (Santa Cruz-58886, Santa Cruz, CA, USA) using the methods described above to provide a loading control. Native signaling proteins were normalized to β-tubulin for comparison.
One milliliter of blood samples was collected from the femoral vein at the time of FHL dissection. The samples were placed in a 1.5-mL microfuge tube, and blood was allowed to coagulate at room temperature for 15 minutes. Serum was separated from the clot by centrifugation at 10,000g for 5 minutes. The serum was placed in a fresh tube and stored at −80°C until analysis. Cortisol was quantified using the Parameter cortisol assay kit (R&D Systems, Inc., Minneapolis, MN, USA) following the procedures recommended by the manufacturer.
Changes in body weight over time were analyzed using repeated-measures multivariate analysis of variance (MANOVA) with grouping variables being the experimental assignment and time. The average fiber CSA was determined for each animal according to fiber type. At least 100 fibers per section (8 sections) were analyzed for each animal. Two-way ANOVA was used for comparisons of fiber CSA and distribution, with grouping variables being the experimental assignment and MyHC isoform expression. Data were analyzed for normality. No outlier exclusion was necessary. One-way ANOVA was used for comparisons of FHL muscle mass, serum cortisol levels, and protein levels. These measurements were performed in triplicate, with coefficient of variance <10%. Post hoc analyses were conducted using the Tukey-Kramer honestly significant difference test as appropriate. JMP software (version 8.0, SAS Inc.) was used for all comparisons. All data are reported as mean ± SE, unless otherwise specified. A p ≤ 0.05 was considered significant.
Training Effect on Body Weight
After a 10-week PRE regimen, there was a significant group and time interaction for the changes in animal body weight (p = 0.02). After 4 weeks, a significant difference in body weight was evident across groups, and by 10 weeks, age-matched SED animals weighed 363 ± 7 g compared with 325 ± 5 g for the PRE group (∼12% difference; p < 0.05). Serum cortisol levels were not significantly different between groups (93 ± 8 vs. 81 ± 11 ng·mL−1 in the PRE and SED groups, respectively), suggesting that hormonal influences or stress levels did not differ across groups.
Effect of Resistance Training on Muscle Hypertrophy (Cross-Sectional Area)
The FHL muscle comprises fibers expressing all 4 MyHC isoforms (Figure 2), with fibers expressing MyHC2X and MyHC2A present in a greater proportion than those expressing MyHC2B or MyHCSlow (Table 1). Overall, the fibers in the FHL muscle were of a larger CSA in the PRE animals (1,714 ± 125 μm2) compared with that in the SED animals (1,512 ± 66 μm2, p = 0.01) (Figure 3). There were differences in fiber CSA according to fiber type (2-way ANOVA; p < 0.001) and no group vs. fiber type interaction (p = 0.69). Overall, the CSA of FHL fibers expressing MyHCSlow, MyHC2A, MyHC2B, and MyHC2X was 10–20% greater in PRE vs. SED animals (Table 1), although only overall differences were statistically significant (Figure 3). Importantly, there were no significant differences in the proportion of fibers expressing different MyHC isoforms after 10 weeks of PRE when compared with that in the SED controls indicative of normal postnatal growth in both groups. Overall, approximately 80% of FHL fibers expressed MyHC2X or MyHC2A independent of group assignment.
Effect of Resistance Training on Myosin Heavy Chain Content
After PRE, there were significant group differences in MyHC content (2-way ANOVA, p = 0.003; with no group vs. fiber type interaction, p = 0.26). Total MyHC content increased by 35% (p < 0.05) in the PRE compared with that in the SED animals (Figure 4). The expression of MyHC2X increased significantly (∼20%; p < 0.05), and although MyHC2A expression increased by 7%, this difference was not statistically significant (Figure 4). Consistent with analyses of fiber type distribution in the FHL muscle (Table 1), MyHC isoforms MyHC2X and MyHC2A comprised approximately 90% of the total MyHC. Both MyHCSlow and MyHC2B isoforms (<10% each) were close to the limit of detection of the protein analysis method, precluding comparisons of these isoforms between groups.
Effect of Resistance Training on Translational Regulatory Proteins
After 10 weeks of training, mTOR, Akt, and AMPK protein levels were expressed equivalently across the PRE and SED groups (Figure 5). Phosphorylation of specific serine (ser) or threonine (thr) residues of mTOR (ser-2481), Akt (ser-473), and AMPK (thr-172) were also not different between groups. Accordingly, when protein quantities were expressed as ratios of phosphorylated to total protein levels, relative phosphorylation levels of mTOR, Akt, and AMPK did not differ between groups (Figure 5). However, the ratio of phosphorylated-to-unphosphorylated rpS6 protein was reduced > sixfold in PRE animals (p < 0.05). Total rpS6 protein levels were unchanged between groups (p > 0.05).
In agreement with our hypotheses, both FHL fiber CSA and MyHC contents were increased after a moderate intensity PRE training program. In this study, we observed a 10–20% increase in the FHL fiber CSA when adolescent animals were trained to carry approximately 80% above their body weight over 10 weeks. These results are generally consistent with those of previous studies using similar models of PRE in rats, but several important methodological differences must be considered. For instance, Hornberger and Farrar (12) investigated FHL adaptations after 8 weeks of progressive ladder climbing, beginning PRE training with mature, adult rats (∼370 g). They indirectly calculated a 17% increase in CSA based on muscle weight and estimated fiber length, similar to our direct measurements of CSA in individual fibers types. By beginning PRE with mature adult animals (∼370 g), Hornberger and Farrar (12) were able to increase loading faster and achieved up to 332% of the body weight after 8 weeks of PRE. The similar gains in CSA with heavier loading may relate to the differences in animal age and weight and to the response to training. Using a climbing apparatus and tail weights similar to that in our experiments, Yarasheski et al. (31) reported an approximately 15% increase in fiber CSA across muscles after an 8-week PRE program in 3-week-old rats that achieved loads of up to 250% above the body weight. It is noteworthy that these findings were similar in different fiber types, with an approximately 13% increase in CSA for rectus femoris fibers classified as type IIb (expressing MyHC2B) and an approximately 16% increase in type IIa fibers (expressing MyHC2A) in the PRE animals compared with that in controls. Duncan et al. (5) implemented a progressive PRE protocol for 26 weeks using 3-week-old rats, and these rats progressed to carrying loads equivalent to 140% of their final body weight. They reported a similar increase in fiber CSA across muscle fibers of different succinate dehydrogenase enzyme (SDH) activity. Low SDH activity fibers of the trained group increased 21% in the extensor digitorum longus, 37% in the plantaris, and 37% in the rectus femoris muscle. Fibers with high and intermediate SDH activity in the soleus muscle of trained animals displayed a 23 and 59% increase in the CSA, respectively.
Although several studies have investigated the physiological adaptations to muscle hypertrophy in response to high-intensity PRE (5,12,31), these studies did not examine the intracellular pathways implicated in muscle hypertrophy (e.g., translational regulators such as rpS6). After 10 weeks of PRE, we investigated sustained changes in the expression and phosphorylation of key signaling molecules that are identified regulators of hypertrophy in adult animal models. The Akt-mTOR signaling molecules and their downstream effects have been implicated as critical regulators of protein synthesis and hypertrophy in varying models of muscle hypertrophy including synergist ablation, growth factor treatment, and mechanical loading in adults (2,13,22,25). However, few if any animal models have explored signaling during hypertrophy in adolescent animals using PRE. It is unclear if hypertrophy in adolescents induced by PRE also depends on Akt-mTOR signaling cascades.
In adolescent animals, we hypothesized that sustained elevations in mTOR, Akt, and rpS6 protein expression may occur as an adaptive response because PRE-induced hypertrophy would occur simultaneously with sustained, rapid growth rates. This is reasonable given that mTOR mRNA levels increase with chronic PRE (16) and that by increasing the expression of constitutively active Akt protein, muscle hypertrophy is significantly increased (2). However, we observed that critical regulators of protein synthesis and translational events—Akt and mTOR—were not elevated in terms of protein expression or phosphorylation levels in the adolescent rat FHL muscle after PRE. These results are consistent with those of prior investigations of chronic PRE and long-term adaptation observed in adult animals (14,32).
Both mTOR and Akt are upstream activators of translational regulators such as p70 S6-kinase, an enzyme that phosphorylates rpS6 (4). The rpS6 protein is a component of the early translation initiation complex used in the synthesis of new proteins and is a suspected downstream candidate for mTOR-induced hypertrophy (27). In contrast to our hypothesis, we observed a reduction in rpS6 phosphorylation levels and no change in rpS6 expression levels. Thus, the current results indicate that chronic elevations in the expression or the phosphorylation of translational regulators, such as rpS6, are not evident for FHL hypertrophy during PRE in rapidly growing adolescents. We speculate that in adolescent animals, mTOR, Akt, and rpS6 response to PRE may be muted in the presence of a chronic state of rapid muscle growth. Postnatal growth of skeletal muscle fibers is substantial in young adolescent animals with fractional protein synthesis rate approximately 2–3 times greater in 3-week-old animals compared with that in 44- and 105-week-old animals, respectively (18). This may be because of naturally rising levels of testosterone and insulin-like growth factor-1 (26).
In chronically trained adults, rpS6 signals may also become muted over time. Karagounis et al. have observed that in adults the acute rpS6 phosphorylation response decreases with repeated bouts of resistance exercise (14). Importantly, in this study, the animals were exercised 3 times over a 5-day period with a day of rest between exercise bouts, and rpS6 phosphorylation was elevated 3 hours after the first and second bouts of resistance exercise but not following the third bout of exercise. Thus, rpS6 signaling likely desensitizes rapidly after repeated bouts of exercise and may be regulated independently of mTOR and Akt. Thus, it appears that rpS6 is activated immediately after acute bouts of PRE in naive adult animals but that the persistence of translational signals is limited with chronic training (14). Therefore, these results suggest that muscle cell signaling pathways may change over time in response to chronic PRE. We speculate that in adolescent animals, rpS6 activity may be muted in the presence of a chronic state of rapid muscle growth. Thus, rpS6 signaling likely desensitizes rapidly after repeated bouts of exercise and may be regulated independently of mTOR and Akt. Genetic studies add complexity to the issue because fibroblasts derived from mutant mice lacking rpS6 exhibit enhanced rates of protein synthesis (28). The role of rpS6 in the regulation of sustained global protein synthesis is thus unclear, and it is suggested that rpS6 is a fine tuner of global protein synthesis with both positive and negative regulation of protein synthesis and growth (27). Interestingly, mTOR and Akt phosphorylation are muted at basal levels with repeated bouts of activity as well (14).
AMP kinase is an important regulator of energy metabolism and has been shown to inhibit mTOR phosphorylation (15,20,21). The AMPK signaling may increase with physical activity (11), which could potentially inhibit mTOR; therefore, we also investigated AMPK phosphorylation levels. We observed that both AMPK protein and phosphorylation levels were unchanged between the PRE and SED groups. The lack of a detectable change in AMPK signaling after PRE is consistent with its primary role as a metabolic sensor (23) likely involved in the regulation of the response to aerobic exercise, not PRE. Alternatively, high metabolic rates in growing adolescent animals may obscure any additional stimuli imposed by PRE.
This study highlights that adolescents are capable of PRE-induced muscle hypertrophy with moderate loading. These results are consistent with the strength gains observed with high-repetition, moderate intensity PRE in children (7). This suggests that lower intensity PRE protocols are sufficient to increase muscle hypertrophy above normal growth rates in adolescents, which has generally been dismissed in the past by indirect studies (26). Because high-repetition, moderate intensity loading is considered safer for children because of lower loading stress on joints, tendon, and muscle, the coach and personal trainer may prefer moderate intensity PRE for preventing injury in adolescent populations. This study prompts the need to readdress whether PRE-induced muscle hypertrophy is an underlying mechanism of strength gain in adolescent children.
The authors thank Ms. Yun-Hua Fang for her technical assistance with immunohistochemistry and Thomas Keller and Rebecca L. Macken for technical assistance with MyHC content analyses. This study was supported by NIH grant R01 AR51173 and the Mayo Clinic.
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